Microtomes are precision mechanical knives used to cut extremely thin sections of specimens for microscopic study. In light microscopy (LM), thin sections are required to produce a specimen conforming with the thin depth of field offered at high magnification by optical instruments. Thin sections also aid in the transmission of light through the specimen to be received by the objective lens of the microscope. Sections may be approximately 1 to 100 microns thick and about 30 mm wide.
Thin sections may also be prepared for electron microscopy. With transmission electron microscopy (TEM), the thin sections aid in the transmission of a sufficient flux of electrons through the specimen to form the image. In the case of scanning electron microscopy (SEM) where depth of field is greater and no transmission of electrons is required, a section or series of sections may nevertheless be used to reveal internal structure. Sections for electron microscopy are prepared with an "ultramicrotome" and may be approximately 40 nm thick and approximately 0.5 mm wide.
In either case, with either a microtome or an ultramicrotome, the samples are normally first embedded in a supporting matrix, impregnated with a supporting material such as a hard plastic, or frozen to make sectioning easier. The prepared sample is then placed in a collet which moves up and down across a microtome knife, the collet advancing toward the cutting plane of the knife after each pass, the amount of advance determining the section thickness.
For light microscopy, the microtome knife may be the sharpened edge of a steel plate, (a "histology" knife) or a fractured edge of a glass plate of similar dimension, (a Ralph knife). For electron microscopy, glass, diamond, and sapphire knives are used.
The extremely slim sections produced by an ultramicrotome require careful handling. In order to facilitate removal of these sections from the knife, it is known in the art to construct a boat that provides a liquid filled trough fitting against the side of the knife from which the section will be dislodged. The liquid in the boat, which may be water or a solvent such as alcohol or acetone, causes the section to float on its surface as a result of buoyancy or surface tension, adhering by one edge to the knife or the previous section. From here, the section may be removed by a fine brush or the like, having been prevented from adhering over its entire surface to the knife blade itself. For TEM, the section is picked up directly by the TEM grid.
With a carefully designed boat, a multiple series of sections can be cut which form a floating chain on the surface of the liquid that may be removed in one piece so as to provide a set of sections of known relationship to each other.
The more expensive knives for ultramicrotomy, such as diamond knives, come glued to a fixture that includes a boat. These knives are relatively long-lived and hence a permanently attached boat is practical.
For glass knives, boats are constructed as they are needed, by the operator of the microtome, using materials at hand. The glass knives used with electron microscopy are generally triangular plates where the width of the plate at one vertex provides the cutting edge. A boat may be formed for these knives by loosely wrapping a single turn of an aluminized Mylar tape about the cutting apex. Gaps may be filled with wax or lacquer. The tape must be trimmed carefully around the knife blade so as not to interfere with the operation of the microtome and without damaging the extremely precise edge of the knife. The construction of such boats is not only time consuming but often the end product is irregular in dimension and produces a liquid surface that pulls the sections to one side where they adhere to the wall of the boat.
These problems are addressed in part by commercially available plastic boats sized to abut one face of the knife and to be attached to the knife with lacquer or wax. Such boats provide a uniform and symmetrical liquid surface on which to receive the sections. Nevertheless, such boats are time consuming to install. The adhesives commonly used to attach such boats to the knives can contaminate the liquid contained in the boat adversely affecting, for example, the way the sections take stain. Normally, the adhesive prevents the boats from being removed for reuse.
Boats are not normally used with the histology or Ralph knives of microtomes but rather these knives are manually wetted with a water or a water-alcohol mixture to minimize adhesion of the section to the knife and to aid in removal of the individual sections as they are cut.